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Background: The immune checkpoint PD-1 and its ligand PD-L1 are involved in the induction of immunological tolerance of solid tumors including oral squamous cell carcinoma (OSCC). The aim of the study was to establish the clinical and prognostic significance of PD-L1 in OSCC.Methods: Tissue microarrays of 125 resected OSCC were stained with two different commercially available PD-L1 antibodies (clones E1L3N and 22C3), alongside PD-1 immunostaining.Results: PD-L1 expression in more than 10% of tumor cells was associated with poorer survival, and established as a clinically relevant cut-off point. This relevant PD-L1 expression was detected in 10% to 15% OSCC specimens depending on the anti-PD-L1 antibody, and showed an inverse correlation with tobacco and alcohol consumption. We consistently found that PD-L1 expression was associated with tumor recurrence and lower disease-specific survival. Multivariate analysis further revealed that neck node metastasis (HR 2.304; P ¼ 0.009) and tumor PD-L1 expression (HR 2.571; P ¼ 0.01) were significant independent factors for poor prognosis.Conclusions: PD-L1 expression in more than 10% of tumor cells was a significant and independent factor of poor prognosis in OSCC.Impact: PD-L1 expression in more than 10% of tumor cells was consistently established as a clinically relevant cut-off point by using two different antibodies. Remarkably, PD-L1 expression emerges as an independent poor prognosis marker in patients with OSCC.

Podoplanin is involved in actin remodeling of the cytoskeleton of tumor cells and may promote tumor cell invasion by increasing cell motility and formation of filopodia-like membrane protrusions. Podoplanin is expressed in a variety of tumors, but its role in head and neck cancer, particularly in oral squamous cell carcinoma, remains unclear. We studied podoplanin expression by immunohistochemistry in 92 oral squamous cell carcinomas (OSCC) using a monoclonal antibody against an epitope of podoplanin (D2-40). In terms of the number of stained cells, 34 OSCC (38 %) had low podoplanin expression (less than 33 % of cells), 33 (36 %) showed moderate expression (between 34 and 66 % of cells), and 21 (22 %) showed high expression. The intensity of immunostaining was strong in 26 (28 %) cases, moderate in 36 (40 %), and weak or negative in the remaining 30 tumors (32 %). Immunohistochemical expression of podoplanin was associated with a tumor histological grade. A diffuse pattern of podoplanin expression significantly decreased in moderately differentiated (37 %) and poorly differentiated (20 %) carcinomas compared to well-differentiated (43 %) carcinomas. In addition, the focal expression of podoplanin in the invasion front of the tumor, without expression in the tumor center, was observed in 72 % of well-differentiated tumors, 27 % of moderate tumors, and 0 % of poorly differentiated tumors. Moreover, a trend was found toward an association of diffuse podoplanin staining with the development of second primary carcinomas (13 %), in contrast to its expression in the invasion front (3 %). No association was observed between podoplanin expression and nodal metastasis.

NANOG, a key regulator of pluripotency and self-renewal in embryonic and adult stem cells, is frequently overexpressed in multiple cancers, including oral squamous cell carcinoma (OSCC). It has been frequently associated with poor outcomes in epithelial cancers, and recently implicated in laryngeal tumorigenesis. On this basis, we investigated the role of NANOG protein expression as an early cancer risk biomarker in oral potentially malignant disorders (OPMD), and the impact on prognosis and disease outcomes in OSCC patients. NANOG expression was evaluated by immunohistochemistry in 55 patients with oral epithelial dysplasia, and 125 OSCC patients. Correlations with clinical and follow-up data were assessed. Nuclear NANOG expression was detected in 2 (3.6%) and cytoplasmic NANOG expression in 9 (16.4%) oral dysplasias. NANOG expression increased with the grade of dysplasia. Cytoplasmic NANOG expression and the histopathological grading were significantly correlated with oral cancer risk, although dysplasia grading was the only significant independent predictor of oral cancer development in multivariate analyses. Cytoplasmic NANOG expression was also detected in 39 (31%) OSCC samples. Positive NANOG expression was significantly associated with tobacco and alcohol consumption, and was more frequent in pN0 tumors, early I-II stages. These data unveil the clinical relevance of NANOG in early stages of OSCC tumorigenesis rather than in advanced neoplastic disease. NANOG expression emerges as an early predictor of oral cancer risk in patients with OPMD.

Potentially malignant oral lesions, mainly leukoplakia, are common. Malignant transformation varies widely, even in the absence of histological features such as dysplasia. Hence, there is a need for novel biomarker-based systems to more accurately predict the risk of cancer progression. The pluripotency transcription factor SOX2 is frequently overexpressed in cancers, including oral squamous cell carcinoma (OSCC), thereby providing a link between malignancy and stemness. This study investigates the clinical relevance of SOX2 protein expression in early stages of oral carcinogenesis as a cancer risk biomarker, and also its impact on prognosis and disease outcome at late stages of OSCC progression. SOX2 expression was evaluated by immunohistochemistry in 55 patients with oral epithelial dysplasia, and in 125 patients with OSCC, and correlated with clinicopathological data and outcomes. Nuclear SOX2 expression was detected in four (7%) cases of oral epithelial dysplasia, using a cut-off of 10% stained nuclei, and in 16 (29%) cases when any positive nuclei was evaluated. Univariate analysis showed that SOX2 expression and histopathological grading were significantly associated with oral cancer risk; and both were found to be significant independent predictors in the multivariate analysis. Nuclear SOX2 expression was also found in 49 (39%) OSCC cases, was more frequent in early tumor stages and N0 cases, and was associated with a better survival. In conclusion, SOX2 expression emerges as an independent predictor of oral cancer risk in patients with oral leukoplakia. These findings underscore the relevant role of SOX2 in early oral tumorigenesis rather than in tumor progression.

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Tumor-associated macrophages (TAMs) can be polarized into antitumoral M1 and protumoral and immunosuppressive M2 macrophages. This study investigated the clinical relevance of TAM infiltration in oral squamous cell carcinoma (OSCC), evaluating CD68 (M1 and M2 macrophage marker) and CD163 expression (M2 macrophage marker) in the tumor nests and surrounding stroma. Immunohistochemical analysis of both stromal/tumoral CD68+ and CD163+ TAMs was performed in paraffin-embedded tissue specimens from 125 OSCC patients, and correlated with clinical data. Potential relationships with the expression of cancer stem cell (CSC) markers and PD-L1 in the tumors were also assessed. Stromal CD163+ infiltration was significantly associated with the tumor location in the tongue, and stromal and tumoral CD68+ and CD163+-infiltrating TAMs were more abundant in nonsmokers and non-alcohol-drinkers. Strikingly, this study uncovers an inverse relationship between CD68+ and CD163+ TAMs and CSC marker expression (NANOG and SOX2) in OSCC. High infiltration of CD163+ TAMs in both tumor and stroma was strongly and significantly correlated with the absence of NANOG expression. Moreover, infiltration of both CD68+ and CD163+ TAMs was also significantly associated with high tumor expression of PD-L1. Our results suggest that there is a link between TAM infiltration and immune escape in OSCC.